Abstract Borrelia burgdorferi is the causative agent of Lyme disease, which affects an estimated 300,000 people annually in the US. When treated early, the disease is usually easily treated with short courses of antibiotics. However, if allowed to progress to late stage symptoms such as arthritis or encephalopathy, longer courses of up to 28 days of antibiotics are recommended. Even with longer courses of antibiotics, a significant proportion of patients with Lyme arthritis will not improve and will require additional courses of treatment. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease. In a preliminary study, we find that B. burgdorferi forms drug-tolerant persister cells similar to those formed by other pathogens. In a pilot Tn-seq experiment, we identified candidate genes involved in persister formation in B. burgdorferi treated with ceftriaxone. The goal of this project is to identify the mechanisms of persister formation in Borrelia burgdorferi, which will help understand their role in drug tolerance. Studying E. coli as a model organism, we identified toxin/antitoxin (TA) modules as a major contributor to formation of dormant persister cells. However, B. burgdorferi lacks TA modules, suggesting that it evolved independent mechanisms of drug tolerance. We developed several complimentary methods to identify persister genes, and these will be applied to the study of B. burgdorferi. First, we will expand our initial identification of mutations that affect persister formation using Tn-seq with additional clinically relevant antibiotics. Transcriptome analysis will be performed on persisters isolated by lysing the culture with a cell wall acting antibiotic; and by sorting persisters following a metabolic marker. High-persister mutants (hip) will be obtained by selection in vitro, and the mutant genes identified by whole genome sequencing. We will also screen for hip mutants from wild ticks and patients to understand if these could account for variations in disease manifestation and response to antibiotics. Priority will be given to candidates that are identified by different approaches. Opposite effects of deletion vs. overexpression of a gene of interest on drug tolerance will validate a persister gene. A detailed examination of the genes will lead to a comprehensive understanding of the mechanism of persister formation and drug tolerance in this pathogen, informing development of better treatments.